The Na-K-Cl cotransporter plays a vital role in transepithelial salt transport and in regulation of blood pressure. The Na-K-Cl cotransporters, NKCC1 and NKCC2, are key elements of net transport in the mammalian renal epithelium: NKCC1 forms part of the final pathway of K+ secretion in the collecting duct, and NKCC2 is key in the process of NaCl reabsorption across the in the thick ascending limb of the loop of Henle (TAL) and distal tubule, where it is the site of action of diuretics such as furosemide. The goal of this project is to understand the mechanisms that underlie the function and regulation of the Na-K-Cl cotransporters in the mammalian kidney. The proposed studies will be carried out with isolated renal tubules as well as with recombinant NKCC1 and NKCC2 protein expressed in the HEK cell expression system. Specifically: 1) We will elucidate the molecular structure of NKCC1, directly addressing ion and inhibitor sites in the translocation pore; we will discover the functional consequence of newly discovered rare human mutations in NKCC1 and NKCC2; and we will examine regulation of NKCC1 in the collecting duct during flow-activated K+ secretion. 2) We will determine the molecular mechanism of NKCC2, focusing on the role that TM2 plays in determination of ion binding kinetics and stoichiometry, and we will discover if intracellular [Cl-] is the signal underlying NKCC2 regulation in the TAL. 3) We will determine the molecular mechanism by which phosphorylation in the NKCC N-termini regulate transport conformational change in the transporter.